Method for treating or preventing dry eyes
a technology for treating or preventing dry eyes, applied in the direction of drug compositions, pharmaceutical delivery mechanisms, medical preparations, etc., can solve the problems of eye swelling, increased risk of toxic and irritation effects, and increased vision damage, so as to increase the endogenous ngf secretion of the lacrimal tissue
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example 1
Compound of Formula (I) on BAC Induced Damage in HCE Cells
[0037]Benzalkonium Chloride Cell Damage Model
[0038]The Human Corneal Epithelial (HCE) cells were seeded on a 6-well plate 5×105 cells with 10% FCS in DMEM / F12 for 24 hours. After cell attachment, medium was removed and changed into 0.0005% Benzalkonium chloride for 24 hours, then washed with PBS and treat with different concentrations 0.4, 2, 10 μg / ml of compound of Formula (I) (compound A) for 48 hours. At the end the cells were trypsinized and centrifuge 1000 rpm for 5 minutes, the supernatant was removed, added 1 ml of DMEM / F12 medium to resuspend the cells, followed by 0.4% trypan blue and the cell culture medium were 1:3 dilution, the number of cells were calculate under a microscope to observe the survivability of different concentrations treatment. The results are shown in FIG. 1. *P<0.05, **P<0.01 compared to the 0 μg / ml compound A group, Student's t-test. Data are expressed as mean±SD (n=3).
example 2
of Formula (I) Improves Tear Secretion in BAC Induced Dry Eye Mice
[0039]Benzalkonium Chloride Induced Dry Eye Mice Model (1)
[0040]Male BALB / c mice (6 weeks) was treated with thrice-daily (9:00, 13:30, 18:00) topical administration of 5 ul of 0.3% BAC in right eyes, mice was treated with PBS as control group, from day 1 to 7. And compound A (2, 5, 10 μg / ml) was treated with thrice-daily (9:00, 13:30, 18:00) topical administration of 5 ul in right eyes, and PBS as control group, from day 8 to 19.
[0041]Phenol red thread tear test was used. The thread is yellow (acidic) and when it comes in contact with tears it changes to a light red color. Forceps were used to insert folded portion of the thread into the palpebral conjunctiva of the eye ⅓ of the distance from the lateral canthus of the lower eye lid. After 15 s, the thread was removed and the entire wet (Red) portion was measured. The phenol red thread test was on 7, 11, 15, 19 day. The results are shown in FIG. 2. *P<0.05, **P<0.01 c...
example 3
Compound of Formula (I) on Tear NGF Protein Expression in BAC Induced Dry Eye Mice
[0045]Tear was collected from right eye of BAC induced dry eye mice by 10 μl PBS wash. Mice treated eyes drop of PBS as control and different concentration Compound A for 10 days. NGF protein was a positive control. 5 μl tear volume used in western blot.
[0046]The total protein concentration was determined by 1×Braford assay. To denature, use a loading buffer(7 ml 0.5M Tris pH6.8, 3 ml Glycerol, 1 g 10% SDS, 0.93 g DTT, 0.5 mg 0.05% bromophenol blue, 10 ml), and boil the mixture at 95-100° C. for 5 minutes. Total protein was loaded 20-40 μg per mini-gel (12% SDS-polyacrylamide gel) well for electrophoresis. Then transfer the proteins into polyvinylidene difluoride membrane and blocking the membrane with 5-10% nonfat dried milk in TTBS. The membrane was incubated with the primary antibody overnight at 4° C. by shaking. Wash the membrane several times in TTBS to remove residual primary antibody. And the m...
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